Instrumental comparison of the determination of Cr3+ uptake by human transferrin

Abstract

UV-VIS absorbance, inductively coupled plasma-optical emission spectroscopy (ICP-OES), and particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) are presented as techniques for determining Cr³⁺ loading into transferrin (Tf), with and without Fe³⁺. The methods are compared based on loading percentages (i.e. 100% loading would be equal to 2 Mⁿ⁺ : 1 Tf) determined for Cr³⁺ loading into apo-transferrin. Spectral interferences and overlapping LMCT bands cause inaccurate chromium (qualitative) and iron (qualitative and quantitative) results for the UV-VIS absorbance method. The ICP-OES and PB/HC-OES methods are in good agreement providing evidence that the PB/HC-OES method is a valid technique for investigating metal–protein complexes. Maximum Cr³⁺ loading into apo-transferrin over a 24 h period was determined to be 26.8 ± 3.5% by the ICP-OES method and 25.3 ± 2.2% by the PB/HC-OES method. Loading percentages were increased to 49.7 ± 1.9% (ICP-OES) and 55.7 ± 3.2% (PB/HC-OES) when the metal-transferrin solution was allowed to incubate for up to 10 days. Under non-excess carbonate conditions the Cr³⁺ loading is elevated over a 24 h incubation time, but under physiological conditions the loading is inhibited. Equal loading of Fe³⁺ and Cr³⁺ into apo-transferrin was achieved when chromium was at a level more than 5 times in excess of iron. Inhibition of Cr³⁺ loading was only observed when an excess of Fe³⁺ was available to bind into apo-transferrin. The ability for Cr³⁺ to displace Fe³⁺ from holo-transferrin was observed as small amounts of Cr³⁺ were loaded into the once occupied metal binding site.